Pex11 proteins were first identified in yeast as peroxisomal membrane proteins that could increase peroxisome number when overexpressed and significantly reduce peroxisome number when interrupted . Early studies suggested that Pex11 proteins acted primarily on medium-chain fatty acid oxidation, affecting peroxisome divisions indirectly . Schrader and colleagues were the first to show in human fibroblasts that overexpression of human Pex11β was sufficient to induce peroxisome proliferation . Recently, it has been shown that Pex11β participates in peroxisome divisions through membrane elongation and shape changes of existing peroxisomes. Elongated membranes fill with imported matrix proteins, form into small blebs and separate into new peroxisomes with the aid of dynamin-like protein . While yeast studies have shown that peroxisomes only arise through division , and mammalian cell studies have suggested that they arise from both de novo and division mechanisms , little is known about peroxisome biogenesis during embryonic development. The question of peroxisome inheritance remains largely unresolved, particularly as we have shown that peroxisomes are absent in early frog embryos, and arise only later due to embryonic and or metabolic cues .
We tested whether overexpression of Pex11β could induce an early-onset to peroxisome biogenesis or accumulation during early Xenopus embryogenesis. This is particularly intriguing, as stage 10 embryos have no detectable peroxisomes. Thus, as Pex11β participates in peroxisome division, and no detectable peroxisomes are present in early embryos, Xenopus represents a novel model where the role of Pex11β in peroxisome number can be examined. The utility of microinjection and relative ease of expression and localization assays enables specific questions related to Pex11β to be addressed. First, we sought to show that Pex11β is sufficient to regulate peroxisome related protein and RNA levels, and increase the number of peroxisomes in X. laevis A6 cells. Our RT-PCR analysis indicated significant increases in RNA levels for both catalase and PMP70, amongst other genes, following overexpression of Pex11β. Using Western blot analysis we confirmed that HA-Pex11β increased catalase and PMP70 proteins levels, and immunohistochemistry confirmed that HA-Pex11β increased the number of both catalase and PMP70 positive punctate structures in A6 cells. Additionally, as GFP-SKL can be transported into peroxisomes, co-transfection of HA-Pex11β and GFP-SKL revealed an increase in the number of peroxisome-like structures. These results strongly support the idea that Pex11β can independently promote increases to the number of peroxisomes in Xenopus A6 cells.
The primary focus of our study was to elucidate the role of Pex11β in vivo. Very little is known about what cellular mechanisms regulate the de novo biogenesis of peroxisome during Xenopus development. Using a different GFP-KANL reporter, we had previously reported their detection at stage 30 in the ectoderm . Histochemical studies in frog have suggested that yolk protein and lipid metabolism occurs at different stages in different tissues [22, 30]. Interestingly, early yolk metabolism is seen in the newly formed muscles - the somites, but not in the large yolk-filled endodermal cells that are present on the ventral side of the embryo [22, 30]. Here, using HA-Pex11β and other specific assays, we demonstrate that peroxisomes are detectable in somites at stage 20, but not at stage 10.
In agreement with the presence of peroxisome by stage 20, the RNA levels of most peroxisomal genes examined changed temporally during early development. Pex11β Pex3, Catalase, and PMP70 showed increasing trends in expression as development proceeded, peaking stage 30, with cytosolic-bound peroxisomal receptor Pex5 not varying during these stages. This suggested that transcripts are present and increasing towards the eventual onset of peroxisome biogenesis and/or their subsequent proliferation. These changes in Pex3 and Pex11β RNA levels relate well with previous studies that have demonstrated their roles in division . If Pex11β did play a key regulatory role, we next determined how microinjecting HA-Pex11β mRNA would affect the relative levels of key peroxisomal genes. Changes of Pex11β RNA levels simply reflect and confirm the presence on the transfected construct. The Pex11β resulted in the significant increases in RNA levels for catalase and PMP70 at all stages tested (10, 20 and 30). There were also increases in the levels of Pex3 and Pex5 at two of the three stages examined, however, these changes were not as dramatic. From this data, we conclude that Pex11β can play a role in the early induction of these peroxisomal genes. Interestingly, as was examined with Pex11β in A6 cells, PPARα RNA levels increased, PPARγ decreased, and PPARδ was unchanged by ectopic Pex11β in embryos. Given that PPARα has roles in the β-oxidation of fatty acids, PPARγ role in lipid catabolism and adipocyte differentiation, and that while expressed ubiquitously, PPARδ functions remain unclear, the significance of our findings are not known. Furthermore, the relationship between PPARs, other metabolic regulators, yolk utilization and peroxisome numbers certainly bears further investigation.
We focused on the distribution of catalase and PMP70 protein within the somites and found that catalase and PMP70 proteins are first localized as punctate structures suggestive of peroxisomes at stage 20, with no detectable signal at stage 10. To corroborate this immunological finding we microinjected GFP-SKL RNA, whose product could be transported into peroxisomes. Our stage 10 histology sections revealed diffuse signals from GFP, indicating that peroxisomes are not yet present, as the SKL-tagged GFP was not localized. However, we were able to show that GFP-SKL localized to punctate-like structures in the somites at stage 20, indicating that peroxisomes are present at this stage.
With these results in mind, we next tested whether microinjecting HA-Pex11β RNA could induce an early accumulation to the number of peroxisomes. While peroxisomes are present at stage 20, perhaps all needed precursors are present earlier in the embryo and waiting a developmental or metabolic cue to form functional peroxisomes. Following the microinjection of HA-Pex11β, we were able to visualize peroxisome-like structures using GFP-SKL at stage 10. This suggested that needed peroxisomal precursors, including matrix proteins and other division proteins, such as dynamin-like proteins are present. Interestingly, together with the data that showed that HA-Pex11β injections increased the transcription of peroxisomal genes, this suggests that Pex11β is a key regulator of peroxisome onset and proliferation during Xenopus development. For the first time, we are able to show that Pex11β can independently induce an early onset to peroxisome accumulation in vivo.